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US20070135382A1 - Use of peroxisome proliferator-activated receptor gamma (ppary) and/or retinoic acid receptor (rxr) agonists to inhibit platelet functions - Google Patents

Use of peroxisome proliferator-activated receptor gamma (ppary) and/or retinoic acid receptor (rxr) agonists to inhibit platelet functions Download PDF

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US20070135382A1
US20070135382A1 US10/576,824 US57682404A US2007135382A1 US 20070135382 A1 US20070135382 A1 US 20070135382A1 US 57682404 A US57682404 A US 57682404A US 2007135382 A1 US2007135382 A1 US 2007135382A1
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pparγ
agonist
platelets
rxr
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Richard Phipps
Patricia Sime
Neil Blumberg
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University of Rochester
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
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    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
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Definitions

  • the present invention was made, at least in part, with funding received from the National Institutes of Health under grant number RO1 DE11390. The U.S. government may retain certain rights in this invention.
  • the present invention relates generally to the use of PPAR ⁇ , PPAR ⁇ agonists and/or RXR agonists to inhibit platelet functions, including platelet aggregation, release of CD40 ligand, release of thromboxanes, release of prostaglandins, and surface expression of CD40 ligand. Consequently, the present invention further relates to uses of PPAR ⁇ agonists and/or RXR agonists to treat patients for CD40 ligand- or thromboxane-mediated conditions.
  • Platelet activation is central to the formation of thrombus, which precipitates most unstable coronary syndromes.
  • the angiographic severity of coronary stenoses may not predict the occurrence of acute cardiac events, since rupture of atheromatous plaque and subsequent thrombosis in slightly stenosed vessels may underlie many myocardial infarctions.
  • the intact endothelium prevents platelet activation, but intimal injury associated with endothelial denudation and plaque rupture exposes subendothelial collagen and von Willebrand factor, supporting prompt platelet adhesion and activation. Local platelet activation then promotes the recruitment of platelets and the formation of thrombus.
  • Platelet inhibitors have included aspirin, thienopyridines, and glycoprotein IIb/IIIa inhibitors. Although these agents have distinct mechanisms of action, all of them inhibit fibrinogen-dependent platelet-platelet associations.
  • Acute myocardial infarction is commonly diagnosed by measuring markers of cardiac necrosis. These markers reflect the extent of cardiac damage but fail to provide direct information about plaque disruption or platelet activation. Because the outcome of acute coronary syndromes is highly dependent on inflammation and thrombosis, it is possible that measurement of these two processes will allow better assessment of plaque instability.
  • Circulating platelet-monocyte aggregates have been shown to be an early marker of acute myocardial infarction (Furman et al., “Circulating Monocyte-Platelet Aggregates are an Early Marker of Acute Myocardial Infarction,” J. Am. Coll. Cardiol. 38:1002-1006 (2001)) and to contribute to the formation of atherosclerotic lesions (Huo et al., “Circulating Activated Platelets Exacerbate Atherosclerosis in Mice Deficient in Apolipoprotein E,” Nat. Med. 9:61-67 (2003)).
  • platelet-monocyte aggregates can provide useful information about the thrombotic or inflammatory state and can identify patients at high risk for cardiac events, their measurement can be cumbersome.
  • measurement of soluble CD40 ligand also called CD154
  • CD154 an immunomodulator
  • CD40 ligand is a trimeric, transmembrane protein of the tumor necrosis factor family and, together with its receptor CD40, is an important contributor to the inflammatory processes that lead to atherosclerosis and thrombosis (Henn et al., “CD40 Ligand on Activated Platelets Triggers an Inflammatory Reaction of Endothelial Cells,” Nature 391:591-594 (1998)).
  • CD40, CD40 ligand, or both Both CD40 and CD40 ligand have been shown to be present in human atheroma (Schonbeck and Libby P, “CD40 Signaling and Plaque Instability,” Circ. Res. 89:1092-1103 (2001)).
  • CD40 ligand is rapidly translocated to the platelet surface after stimulation and is up-regulated in fresh thrombus (Henn et al., “CD40 Ligand on Activated Platelets Triggers an Inflammatory Reaction of Endothelial Cells,” Nature 391:591-594 (1998)).
  • the surface-expressed CD40 ligand is then cleaved from the platelets over a period of minutes to hours, subsequently generating a soluble fragment (soluble CD40 ligand) (Andre et al., “Platelet-Derived CD40L: The Switch-Hitting Player of Cardiovascular Disease,” Circulation 106:896-899 (2002)).
  • CD40 ligand Although it may also be shed from stimulated lymphocytes, it is estimated that more than 95 percent of circulating CD40 ligand is derived from platelets (Andre et al., “Platelet-Derived CD40L: The Switch-Hitting Player of Cardiovascular Disease,” Circulation 106:896-899 (2002)). Soluble CD40 ligand has been shown to be associated with an increased risk of cardiovascular events in apparently healthy women (Schonbeck et al., “Soluble CD40L and Cardiovascular Risk in Women,” Circulation 104:2266-2268 (2001)).
  • Heeschen et al. provide important information about the clinical relevance of levels of soluble CD40 ligand in patients presenting with chest pain (Heeschen et al., “Soluble CD40 Ligand in Acute Coronary Syndromes,” N. Engl. J. Med. 348:1104-1111 (2003)). In their study, soluble CD40 ligand identified patients at high risk for acute coronary syndromes.
  • Peroxisome proliferator-activated receptors are members of a nuclear hormone receptor superfamily of ligand-activated transcription factors. There are three PPAR subtypes PPAR ⁇ , PPAR ⁇ / ⁇ and PPAR ⁇ . The genes encoding the PPAR subtypes each reside on different chromosomes and have distinct tissue expression patterns (Daynes and Jones, “Emerging Roles of PPARs in Inflammation and Immunity,” Nature Rev. Immunol. 2:748-759 (2002)).
  • PPAR ⁇ While many reports focus on PPAR expression in the nucleus, PPAR ⁇ , in particular, is also found in the cytoplasm (Padilla et al., “Human B Lymphocytes and B Lymphomas Express PPAR- ⁇ and Are Killed by PPAR- ⁇ Agonists,” Clinical Immunology 103:22-33 (2002); Kelly et al., “Commensal Anaerobic Gut Bacteria Attenuate Inflammation by Regulating Nuclear-Cytoplasmic Shuttling of PPAR- ⁇ and Rel A,” Nat. Immunol. 5:104-112 (2004)).
  • PPAR ⁇ is highly expressed in white adipose tissue and was initially described as being important for regulating gene expression in metabolism, insulin responsiveness, and adipocyte differentiation (Spiegelman et al., “PPAR gamma and the Control of Adipogenesis,” Biochemie. 79:111-112 (1997); Fajas et al., “The Organization, Promoter Analysis and Expression of the Human PPAR ⁇ Gene,” J. Biol. Chem. 272:18779-18789 (1997)).
  • PPAR ⁇ was originally thought to be found mainly in fat tissue, it is in fact widely expressed by many types of cells including macrophages, B and T lymphocytes, epithelial, endothelial, smooth muscle, and fibroblastic cells
  • Macrophage Activation a Negative Regulator of Macrophage Activation
  • Harris and Phipps “Prostaglandin D 2 , its Metabolite 15-d-PGJ 2 , and Peroxisome Proliferator Activated Receptor-Gamma Agonists Induce Apoptosis in Transformed, but not Normal, Human T Lineage Cells,” Immunology 105:23
  • PPAR ⁇ has also come to prominence as PPAR ⁇ agonists play an important role in immune function by dampening inflammation, by attenuating macrophage/monocyte synthesis of proinflammatory cytokines, and by inducing apoptosis in B lymphocytes (Jiang et al., “PPAR-Gamma Agonists Inhibit Production of Monocyte Inflammatory Cytokines,” Nature 391:82-86 (1998); Ricote et al., “The Peroxisome Proliferator-activated Receptor-gamma is a Negative Regulator of Macrophage Activation,” Nature 391:79-82 (1998); Padilla et al., “Human B Lymphocytes and B Lymphomas Express PPAR- ⁇ and Are Killed by PPAR- ⁇ Agonists,” Clinical Immunology 103:22-33 (2002); Padilla et al., “Peroxisome Proliferator Activator Receptor- ⁇ Agonists
  • PPAR ⁇ has also emerged as a key target for malignant cells, as PPAR ⁇ agonists have shown therapeutic potential for B lymphoma and various epithelial-derived cancers (Padilla et al., “Human B Lymphocytes and B Lymphomas Express PPAR- ⁇ and Are Killed by PPAR- ⁇ Agonists,” Clinical Immunology 103:22-33 (2002); Jackson et al., “Potential Role for Peroxisome Proliferator Activated Receptor (PPAR) in Preventing Colon Cancer,” Gut 52:1317-1322 (2003); Mueller et al., “Terminal Differentiation of Human Breast Cancer Through PPAR Gamma,” Molecular Cell. 1:465-470 (1998)).
  • Megakaryocytes are the biggest cell of the bone marrow and the parent cell of platelets. Platelets are derived from the cytoplasm of megakaryocytes and are released to the bloodstream under the effects of cytokines such as IL-6 and IL-11 (Teramura et al., “Interleukin-11 Enhances Human Megakaryocytopoiesis in vitro,” Blood 79:327-331 (1992); Burstein et al., “Thrombocytopoiesis in Normal and Sublethally Irradiated Dogs: Response to Human Interleukin-6,” Blood 80:420-428 (1992)).
  • cytokines such as IL-6 and IL-11
  • Platelets are nuclear cells that have a plasma membrane, surface-connected canalicular and tubular system, mitochondria, granules, lysosomes, and peroxisomes (Bentfeld-Barker and Bainton, “Identification of Primary Lysosomes in Human Megakaryocytes and Platelets,” Blood 59:472-481 (1982)). Recent studies demonstrate that platelets and many of their products are not only important in hemostasis, but have now emerged as important in immunoregulation and inflammation.
  • platelets produce key inflammatory mediators such as transforming growth factor- ⁇ (TGF- ⁇ ), thromboxane A 2 , and PGE 2 (Scheuerer et al., “The CXC-chemokine Platelet Factor 4 Promotes Monocyte Survival and Induces Monocyte Differentiation into Macrophages,” Blood 95:1158-1166 (2000); Gear et al., “Platelet Chemokines and Chemokine Receptors: Linking Hemostasis, Inflammation, and Host Defense,” Microcirculation 10:335-350 (2003); Vezza et al., “Prostaglandin E2 Potentiates Platelet Aggregation by Priming Protein Kinase C,” Blood 82:2704-2713 (1993)).
  • CD40L CD40 ligand
  • the present invention relates to the surprising findings that human megakaryocytes and platelets express PPAR ⁇ , and are susceptible to PPAR ⁇ agonists and RXR agonists that dampen proinflammatory and proatherogenic platelet functions, including platelet aggregation, platelet release of CD40 ligand, release of thromboxanes, release of prostaglandins, and surface expression of CD40 ligand.
  • a first aspect of the present invention relates to a method of inhibiting release of CD40 ligand, thromboxanes, or prostaglandin E2, or inhibiting CD40 ligand surface expression, by mammalian platelets.
  • This aspect of the present invention includes the step of contacting mammalian platelets with an effective amount of PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, or a combination thereof, whereby said contacting inhibits release of CD40 ligand, thromboxanes, prostaglandin E2, or a combination thereof, and/or inhibits CD40 ligand surface expression by the mammalian platelets.
  • a second aspect of the present invention relates to a method of inhibiting thrombosis.
  • This aspect of the present invention includes the step of contacting mammalian platelets with an effective amount of PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, or a combination thereof, whereby said contacting inhibits formation of a thrombosis by the mammalian platelets.
  • a third aspect of the present invention relates to a method of treating or preventing a thrombotic condition or disorder.
  • This aspect of the invention includes the step of contacting mammalian platelets, in an individual exhibiting symptoms of or predisposed to a thrombotic condition or disorder, with an effective amount of PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, or a combination thereof, whereby said administering inhibits platelet activation to treat or prevent the thrombotic condition or disorder.
  • a fourth aspect of the present invention relates to a method of improving the quality of a blood product.
  • This aspect of the invention includes the steps of providing PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, an inducer of a PPAR ⁇ agonist, or a combination thereof; and introducing PPAR ⁇ , the PPAR ⁇ agonist, the RXR agonist, the inducer of a PPAR ⁇ agonist, or the combination thereof, to a blood product, wherein the PPAR ⁇ agonist, the RXR agonist, the inducer of a PPAR ⁇ agonist, or the combination thereof inhibits clotting or activation of platelets in the blood product and thereby improves the quality thereof.
  • a fifth aspect of the present invention relates to a stored blood product that includes: a blood product that contains platelets and an amount of PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, an inducer of a PPAR ⁇ agonist, or a combination thereof that is effective to inhibit platelet activation.
  • a sixth aspect of the present invention relates to a method of inhibiting platelet aggregation.
  • This aspect of the present invention includes the step of contacting mammalian platelets with an effective amount of PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, or a combination thereof, whereby said contacting inhibits aggregation of the mammalian platelets.
  • a seventh aspect of the present invention relates to a method of treating or preventing a CD40 ligand-mediated or thromboxane-mediated condition.
  • This aspect of the invention includes the step of contacting platelets, in an individual exhibiting or predisposed to a CD40 ligand-mediated or thromboxane-mediated condition, with PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, an inducer of a PPAR ⁇ agonist, or a combination thereof, whereby said contacting inhibits the release of CD40 ligand and/or thromboxane by platelets, thereby treating or preventing the CD40 ligand-mediated or thromboxane-mediated condition.
  • An eighth aspect of the present invention relates to a method of assessing the activity of a compound as a PPAR ⁇ agonist.
  • This aspect of the invention includes the steps of: combining a compound with both platelets and a platelet activator; determining the level of CD40 ligand or thromboxane released from the platelets; and comparing the level of CD40 ligand or thromboxane released from the platelets to the level of CD40 ligand or thromboxane released from a standard, wherein deviation from the standard, or absence thereof, indicates activity of the compound as a PPAR ⁇ agonist.
  • a ninth aspect of the present invention relates to a method of diagnosing a CD40 ligand-mediated condition.
  • This aspect of the invention includes the steps of: obtaining a patient sample; and determining the level of PPAR ⁇ in the patient sample, wherein a reduced (or lower than normal) PPAR ⁇ level indicates the presence of a CD40 ligand-mediated condition.
  • a tenth aspect of the present invention relates to a method of assessing the efficacy of a PPAR ⁇ agonist therapy.
  • This aspect of the invention includes the steps of: obtaining a patient sample, the patient having been previously administered a PPAR ⁇ agonist or an inducer of a PPAR ⁇ agonist for treating a medical condition or disorder; and determining the level of PPAR ⁇ in the patient sample, wherein an elevated PPAR ⁇ level, relative to a baseline PPAR ⁇ level for the patient prior to said administration, indicates the efficacy of the PPAR ⁇ agonist therapy.
  • An eleventh aspect of the present invention relates to a method of treating or preventing a CD40 ligand-mediated condition.
  • This aspect of the invention includes the step of: treating a patient exhibiting or predisposed to a CD40 ligand-mediated condition with recombinant PPAR ⁇ , whereby said treating inhibits the release of CD40 ligand by platelets, thereby treating or preventing the CD40 ligand-mediated condition.
  • a twelfth aspect of the present invention relates to a method of modifying megakaryocytes.
  • This aspect of the present invention includes the step of exposing a megakaryocyte to PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, an inducer of a PPAR ⁇ agonist, or a combination thereof, whereby said exposing phenotypically modifies the megakaryocyte to produce daughter platelets that minimize pro-inflammatory and/or prothrombotic responses by the platelets.
  • a thirteenth aspect of the present invention relates to a method of treating or preventing a CD40 ligand-mediated or thromboxane-mediated condition.
  • This aspect of the present invention includes the step of treating a patient exhibiting or predisposed to a CD40 ligand-mediated condition with recombinant PPAR ⁇ , whereby said treating inhibits the release of CD40 ligand and/or thromboxane by platelets, thereby treating or preventing the CD40 ligand-mediated or thromboxane-mediated condition.
  • the use of PPAR ⁇ agonist to blunt the release of CD40 ligand or thromboxanes by platelets can effectively control the level of soluble CD40 ligand or thromboxanes that are present in an individual's circulatory system.
  • CD40 ligand, thromboxanes and/or prostaglandins, PPAR ⁇ , the PPAR ⁇ agonist, or inducers of PPAR ⁇ agonist can be used to treat or prevent development of conditions or disorders mediated by CD40 ligand, thromboxanes, or prostanglandins, including thrombotic conditions or disorders.
  • FIGS. 1 A-D demonstrate that PPAR ⁇ protein is expressed in the human megakaryoblast cell line, Meg-01, and by human platelets.
  • FIG. 1A is a Western blot of Meg-01 cell line (15 ⁇ g) using a polyclonal anti-PPAR ⁇ antibody (Calbiochem). PPAR ⁇ bands co-migrated with the human adipose tissue protein extract (15 ⁇ g) used as positive control.
  • FIGS. 1 B-C are Western blots for PPAR ⁇ using platelet cell lysates (15 ⁇ g) from rigorously purified single donor or pooled platelets.
  • FIG. 1B was prepared using monoclonal anti-PPAR ⁇ antibody (Santa Cruz); FIG.
  • FIG. 1C was prepared using a polyclonal anti-PPAR ⁇ antibody (Calbiochem). Human adipose tissue protein extract (5 ⁇ g) was used as positive control (first lanes). The PPAR ⁇ protein was shown for three different single donor and pooled platelets samples. Purified human red blood cells (30 ⁇ g) are negative for PPAR ⁇ ( FIG. 1B ). Data are representative of more than five experiments.
  • FIG. 1D shows that purified mouse platelets (5 ⁇ g) also express PPAR ⁇ (lane 1, adipose tissue; lane 2, mouse macrophage cell line; lanes 3 & 4, purified platelets from two different mice).
  • FIGS. 2 A-D demonstrate PPAR ⁇ expression in human megakaryoblast cells and platelets. Immunocytochemistry was performed with a rabbit polyclonal anti-PPAR ⁇ antibody as described in the Examples. Non-specific staining was assessed using a rabbit IgG isotype control.
  • FIG. 2A shows that nucleated cells and enucleated platelet-like cells of the Meg-01 cell line were stained for PPAR ⁇ . Meg-01 cells stain in the nucleus and the cytoplasm. Results were repeated four times with separate preparations of Meg-01 cells. Original magnification is X600.
  • FIG. 2B shows that human platelets express PPAR ⁇ . The staining pattern for PPAR ⁇ is throughout the platelets.
  • FIG. 2C shows the flow cytometric analysis for intracellular expression of PPAR ⁇ in human platelets. Purified platelets were washed and stained with a monoclonal FITC-labeled anti-PPAR ⁇ antibody (open histogram) or FITC-labeled IgG1 isotype control (shaded histogram) as described in the Examples. Forward and side scatter gates were set to analyze only platelets. This experiment was repeated three times with similar results.
  • FIG. 2D shows the immunocytochemistry of human bone marrow megakaryocyte for PPAR ⁇ . Left panel shows a Diff-Quick staining of a human bone marrow megakaryocyte.
  • Immunohistochemistry was performed with a mouse monoclonal PPAR ⁇ antibody as described in the Examples. PPAR ⁇ expression is shown in the right panel. Mouse IgG1 isotype control was also used to show non-specific staining (middle panel). In addition to PPAR ⁇ immunostaining, light counterstaining was performed with Hematoxylin to visualize the cells. The arrows are pointing at human megakaryocytes. Original magnification is X600. Data are representative of four experiments from four patients with similar results.
  • FIG. 3 is a gel electrophoresis of reverse transcription polymerase chain reaction products, demonstrating that the human megakaryocyte cell line, Meg-01, but not human platelets express PPAR ⁇ mRNA.
  • Total RNA was isolated from Meg-01 cells (lane 6) and human platelets (lanes 3-5) and reverse transcribed into cDNA.
  • the cDNA was amplified with primers specific for ⁇ -actin (539 bp product, as a control) or PPAR ⁇ (360 bp product).
  • a 100 bp ladder was loaded in lane 1.
  • Human adipose tissue (lane 2) and the human monocyte cell line (THP1) (lane 7) were used as positive controls. Platelet samples were from a single donor (lane 3) or pooled from several donors (lanes 4 and 5). Reverse transcriptase ( ⁇ ) controls were negative in all cases.
  • FIGS. 4 A-C demonstrate that Meg-01 cells and human platelets contain PPAR ⁇ that binds the PPAR ⁇ DNA consensus sequence.
  • FIG. 4A shows that 15d-PGJ 2 and ciglitazone induce DNA binding of PPAR ⁇ protein in Meg-01 cells.
  • an electrophoretic mobility shift assay (EMSA) was performed.
  • Lane 1 was loaded with free probe (no lysate), and lane 5 was loaded with nuclear extract from 15d-PGJ 2 treated cells incubated with unlabeled probe (cold competitor) as a control for binding specificity.
  • Lane 6 shows the locations of shifted and supershifted PPAR ⁇ (supershift with an anti-PPAR ⁇ antibody). Shift assays were repeated three times with similar results.
  • FIG. 4B shows via EMSA that platelets have PPAR ⁇ DNA binding activity. Platelet extracts were prepared without any treatment from three different pooled platelets as described in the Examples. Lane 1 shows radioactive-labeled probe. Fifty ⁇ g of cell extracts were incubated with 32 P-labeled PPAR ⁇ oligonucleotides (lane 2, 3, and 4) or cold competitor (unlabeled probe) (lane 5, 6, 7), and then run on a 4% nondenaturing gel. Lanes 8, 9 and 10 indicate the locations of supershifted bands with anti-PPAR ⁇ antibody.
  • FIG. 4C shows via transAM® solid phase PPAR ⁇ DNA binding activity measurements that platelets have some active DNA binding PPAR ⁇ without treatment with PPAR ⁇ agonist.
  • PPAR ⁇ agonist (20 ⁇ M 15d-PGJ 2 , ciglitazone, rosiglitazone) significantly enhances binding to the PPAR ⁇ DNA response element.
  • Assay background in this experiment was 0.02 OD.
  • FIG. 5 shows that human platelets express RXR protein.
  • Rigorously purified human platelets were lysed (5 ⁇ g protein), and their probed with an anti-RXR antibody by western blot. Platelets do express RXR, as shown in lanes 1-3. Human adipose tissue was used as positive control.
  • FIGS. 6 A-B illustrate the effects of PPAR ⁇ agonists on blocking platelet release of CD40L and thromboxane.
  • Purified human platelets were exposed to buffer or with 20 ⁇ M 15d-PGJ 2 or rosiglitazone for 15 minutes. The platelets were then activated with 0.8 U/ml thrombin and the supernatants collected at the times shown.
  • FIG. 7 illustrates the ability of PPAR ⁇ agonists to block the thrombin-induced increase in platelet surface CD40L expression.
  • Purified human platelets were exposed to 20 ⁇ M 15d-PGJ 2 or rosiglitazone for 15 minutes and were then stimulated with 0.8 U/ml thrombin for 60 minutes. The platelets were then stained and prepared for flow cytometry with a monoclonal anti-human CD40L antibody or with control isotype antibody. The graph shows a representative experiment with the results presented as the percent of surface CD40L positive platelets.
  • FIGS. 8 A-B illustrate the effects of PPAR ⁇ agonists on platelet function.
  • ATP release from platelets during aggregation was characterized by Lumi-Aggregometry after stimulation with thrombin (1 U/ml) or ADP (5 ⁇ M). Both the magnitude and rate of ATP release were reduced ( FIG. 8A ).
  • the rate of release with normal platelets was defined as 100%.
  • the results represent mean ⁇ SEM of three experiments.
  • 8B illustrates the results of a typical experiment showing platelet aggregation with normal control (ADP as platelet agonist) (left panel) compared to 15d-PGJ 2 and rosiglitazone (10 ⁇ M) (right panel). Both of these PPAR ⁇ agonists decrease the aggregation slope (rate).
  • FIG. 9 is a graph illustrating the effects of rosiglitazone (Avandia), a PPAR ⁇ agonist, in attenuating the ability of the platelet activator epinephrine to induce a clot.
  • rosiglitazone Avandia
  • PPAR ⁇ agonist a PPAR ⁇ agonist
  • FIG. 10 is a graph illustrating the effects of the PPAR ⁇ agonist 15d-PGJ 2 in attenuating the ability of the platelet activator ADP to induce a clot.
  • FIG. 11 illustrates the effects of PPAR ⁇ agonists on blocking platelet release of prostaglandin E 2 (PGE 2 ).
  • FIGS. 12 A-B illustrate the detection of PPAR ⁇ in human and mouse tissue and fluid samples as detected by Western blot using monoclonal or polyclonal antibodies that react with PPAR ⁇ .
  • FIG. 13 shows the postulated pathways for natural and synthetic PPAR ⁇ small molecule agonists to bind and activate PPAR ⁇ and to regulate transcription. The figure also reveals that there are potential PPAR ⁇ independent pathways whereby PPAR ⁇ agonists can function.
  • PPRE PPAR response element in DNA that leads to gene transcription.
  • FIG. 14 schematically illustrates findings linking Diabetes to PPAR ⁇ , platelet activation, thrombosis and inflammation.
  • the figure shows that human platelets become activated to express surface CD40L, to aggregate, to release CD40L, as well as other proinflammatory and prothrombotic mediators. Note that exposure of platelets to PPAR ⁇ agonists blunts their activation, aggregation and release of proinflammatory and prothrombotic mediators. The end result should be a dampening of thrombosis and inflammation in diabetic and non-diabetic subjects.
  • the present invention generally involves inhibiting mammalian 5 platelet release of CD40 ligand, thromboxanes, and prostaglandin E2, as well as inhibiting platelet surface expression of CD40 ligand, by contacting the mammalian platelets with PPAR ⁇ , a PPAR ⁇ agonist (or inducer thereof), an RXR agonist, or a combination thereof.
  • the present invention allows for reducing platelet activation and therefore inhibiting platelet aggregation and clot formation; inhibiting thrombus formation by activated platelets, as well as treating or preventing CD40 ligand-mediated conditions and/or thromboxane-mediated conditions.
  • the present invention also contemplates inhibiting CD40 ligand release and/or synthesis by megakaryocytes, the cells responsible for production of platelets.
  • the mammalian platelets whose release of CD40 ligand or thromboxane stores can be inhibited, can be any mammalian platelet that expresses PPAR ⁇ .
  • Preferred mammalian platelets are human platelets, although other mammalian platelets, such as those from dogs, cats, horses, cows, pigs, other primates, etc. can also be treated in accordance with the present invention.
  • the mammalian platelets, when contacted in accordance with the present invention can be located in vitro, ex vivo, or in vivo (i.e., in a patient to be treated in accordance with the present invention).
  • the terms “patient” and “individual” refer to any mammal whose platelets contain CD40 ligand or thromboxane stores.
  • the patient or individual to be treated can be a diabetic patient susceptible to CD40-ligand mediated conditions or thromboxane-mediated conditions.
  • the patient or individual to be treated is non-diabetic.
  • the PPAR ⁇ can be either substantially purified PPAR ⁇ (i.e., purified from mammalian tissue) or recombinant PPAR ⁇ .
  • Recombinant human PPAR ⁇ is commercially available from Calbiochem Corp./EMD Biosciences (San Diego, Calif.).
  • fragments thereof that are capable of blunting platelet release of CD40 ligand, thromboxanes, or PGE 2 can also be used. Fragments possessing one or more domains can be used, such as those possessing one or more of the ligand binding domain, DNA binding domain, RXR dimerization domain, or co-activator interacting domain(s).
  • PPAR ⁇ agonists are agents that bind to PPAR ⁇ and activate receptor-activated pathways.
  • the PPAR ⁇ agonists can optionally have dual activity on other PPAR receptors (PPAR ⁇ and PPAR ⁇ ).
  • Exemplary PPAR ⁇ agonists include, without limitation, cyclopentenone class prostaglandins, thiazolidinediones, glitazones, lysophosphatidic acid (“LPA”) or LPA derivatives (McIntyre et al., “Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPAR gamma agonist,” Proc. Natl. Acad. Sci.
  • tyrosine-based agonists indole-derived agonists, and combinations thereof.
  • a preferred member of the cyclopentenone class of prostaglandins is 15D-prostaglandin J 2 .
  • Preferred thiazolidinediones and/or glitazones include, without limitation, ciglitazone, troglitazone, pioglitazone, rosiglitazone, SB213068 (Smith Kline Beecham), GW1929, GW7845 (Glaxo-Wellcome), and L-796449 (Merck).
  • Suitable tyrosine-based agonists include N-(2-benzylphenyl)-L-tyrosine compounds (Henke et al., N-(2-benzylphenyl)-L-tyrosine PPARgamma Agonists: Discovery of a Novel Series of Patent Antihyperglycemic and Antihyperlipidemic Agents,” J. Med. Chem. 41:5020-5036 (1998), which is hereby incorporated by reference in its entirety.
  • Suitable indole-derived agonists include those disclosed, e.g., in Hanks, et al., “Synthesis and Biological Activity of a Novel Series of Indole-derived PPARgamma Agonists,” Biorg. Med. Chem LLH. 9(23):3329-3334 (1999), which is hereby incorporated by reference in its entirety. Any other PPAR ⁇ agonists, whether now known or hereafter developed, can also be utilized in accordance with the present invention.
  • inducers of PPAR ⁇ agonists can also be utilized in accordance with the present invention.
  • Inducers of PPAR ⁇ agonists are agents that induce an increase in the expression or production of a native PPAR ⁇ agonist.
  • Exemplary inducers of PPAR ⁇ agonists include, without limitation, decorin or fragments thereof, enzymes that catalyze formation of prostaglandin D 2 precursor, and combinations thereof.
  • Decorin is a small chondroitin/dermatan sulphate proteoglycan that binds the cytoline transforming growth factor beta (TGF- ⁇ ) through its core protein.
  • Preferred enzymes that catalyze formation of prostaglandin D 2 precursor are hematopoiefic prostaglandin-D synthase and a lipocalin-form prostaglandin-D synthase. Any other inducers of PPAR ⁇ agonists, whether now known or hereafter developed, can also be utilized in accordance with the present invention.
  • RXR agonists are agents that bind to the retinoic acid receptor and activate receptor-activated pathways.
  • exemplary RXR agonists include, without limitation, 9-cis-retinoic acid, trans-retinoic acid, any synthetic RXR agonists, e.g., those available from Ligand Pharmaceuticals (San Diego, Calif.), and combinations thereof. Any other RXR agonists, whether now known or hereafter developed, can also be utilized in accordance with the present invention.
  • PPAR ⁇ or PPAR ⁇ agonists can also be used in combination with the other agents described above.
  • PPAR ⁇ agonists are agents that bind to PPAR ⁇ and activate receptor-activated pathways
  • PPAR ⁇ agonists are agents that bind to PPAR ⁇ and activate receptor-activated pathways.
  • a number of known agonists have dual receptor activity. Any PPAR ⁇ agonists or PPAR ⁇ agonists, whether now known or hereafter developed, can also be utilized in accordance with the present invention.
  • a number of in vitro and ex vivo uses are expected with the present invention, including stored blood products and treatment of blood outside of the body for its immediate return, such as in the case of a dialysis machine or a heart-lung machine.
  • any in vitro or ex vivo activity involving storage or handling of blood products can be enhanced in accordance with the present invention.
  • the stored blood products of the present invention include (i) an amount of PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, an inducer of a PPAR ⁇ agonist, or a combination thereof that is effective to inhibit platelet activation; and (ii) any blood product that contains platelets.
  • the PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, an inducer of a PPAR ⁇ agonist, or a combination thereof is intended to be supplied exogenously. That is, with respect to PPAR ⁇ or naturally occurring inducers of PPAR ⁇ , they are added to the stored blood product in addition to any quantities that may naturally be present therein.
  • the stored blood product can also contain an anticoagulant or other agents, e.g., PPAR ⁇ agonists.
  • Exemplary stored blood products include, without limitation, whole blood, plasma, white blood cell products, and concentrated platelets.
  • This aspect of the present invention generally involves introducing to a blood product either PPAR ⁇ , a PPAR ⁇ agonist, an RXR agonist, or a combination thereof, wherein the introduced agents inhibit clotting or activation of platelets in the blood product and thereby improve the quality thereof.
  • the introducing of the above-identified agents to the blood product is preferably carried out prior to any storage of the blood product.
  • whole blood it is therefore desirable to collect whole blood from a patient or donor into a receptacle that already contains one or more of the above-identified agents.
  • blood fractions such as plasma or concentrated platelets
  • Suitable dosages for in vitro and ex vivo uses include doses between about 1 ⁇ M and about 100 ⁇ M, preferably between about 1 ⁇ M to about 50 ⁇ M, more preferably between about 1 ⁇ M to about 10 ⁇ M.
  • administration of the PPAR ⁇ agonist or the inducer of a PPAR ⁇ agonist to a mammal can be achieved in a manner that achieves a desired reduction in the release of pro-inflammatory and pro-thrombotic modulators, such as CD40 ligand, thromboxanes, and PGE2.
  • pro-inflammatory and pro-thrombotic modulators such as CD40 ligand, thromboxanes, and PGE2.
  • CD40 ligand and/or thromboxane levels are contemplated.
  • routes of administration any one or more routes of administration can be utilized, preferred modes of administration include, without limitation, topical application, intranasal instillation, inhalation, intravenous injection, intra-arterial injection, intramuscular injection, application to a wound site, application to a surgical site, intracavitary injection, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intraplurally, intraperitoneally, intraventricularly, intra-articularly, intra-aurally, intraocularly, or intraspinally.
  • the present invention also affords a method of inhibiting the activation of platelets, inhibiting the aggregation of activated platelets, and inhibiting the formation of clots that contain activated platelets. Consequently, the present invention likewise affords a method of treating or preventing thrombotic conditions or disorders.
  • Thrombotic conditions or disorders to be treated or prevented can include one or more of stroke, venous or arterial thrombosis, disseminated intravascular coagulation, myocardial infarction, pulmonary thrombo-embolism, and pulmonary hypertension (primary or secondary).
  • Any of the above listed agents, including PPAR ⁇ , PPAR ⁇ agonists, RXR agonists, inducers of a PPAR ⁇ agonist, or combinations thereof, can be administered (as noted above) to treat or prevent the thrombotic condition or disorder.
  • Patients to be treated in accordance with this aspect of the present invention can be those exhibiting symptoms of or predisposed to a thrombotic condition or disorder.
  • Symptoms of thrombotic conditions or disorders can include, without limitation, pain, numbness, loss of function, swelling, bleeding, weakness, arrhythmia, pallor, shortness of breath, dysphasia, aphasia, dysarithyia, visual loss, paresis, hearing loss, bruising, and syncope.
  • Persons predisposed to thrombotic conditions or disorders are those patients currently asymptomatic, and can include those having a family history of such conditions or disorders or having had prior treatment for such conditions or disorders.
  • the present invention also affords the treatment or prevention of a CD40 ligand- or thromboxane-mediated conditions.
  • CD40 ligand has been implicated in a number of diseases or disorders including, without limitation, diabetes, atherosclerosis, induced multiple sclerosis, venous or arterial thrombosis, pulmonary fibrosis, systemic lupus erythematous, renal fibrosis, hepatic cirrhosis, cerebral gliosis, disseminated intravascular coagulation, myocardial infarction, pulmonary thrombo-embolism, and pulmonary hypertension.
  • Any PPAR ⁇ agonist, RXR agonist, inducer of a PPAR ⁇ agonist, or combinations thereof can be administered (as noted above) to treat or prevent the CD40 ligand-mediated condition.
  • Patients to be treated in accordance with this aspect of the present invention can be those exhibiting symptoms of or predisposed to CD40 ligand-mediated condition.
  • Symptoms of CD40 ligand-mediated conditions can include, without limitation, shortness of breath, cough, edema or swelling (both generally and particularly in the legs), chest pain, limb weakness, claudication, polyurea, polydipsia, bruising, bleeding, limb pain, and abdominal pain and swelling.
  • Persons predisposed to CD40 ligand-mediated conditions are those patients currently asymptomatic, and can include those having a family history of such conditions or disorders or having had prior treatment for such conditions or disorders.
  • Suitable dosage levels include those capable of achieving blood levels of about 1 ⁇ M up to about 1 mM, preferably about 1 ⁇ M up to about 500 ⁇ M, more preferably about 1 ⁇ M up to about 100 ⁇ M.
  • Thromboxanes A 2 and B 2 have been implicated in a number of diseases or disorders including, without limitation, diseases of coagulation, asthma, anti-phospholipid syndrome, and those involving chronic inflammation (e.g., Rheumatid arthritis).
  • Any PPAR ⁇ agonist, RXR agonist, inducer of a PPAR ⁇ agonist, or combinations thereof can be administered (as noted above) to treat or prevent the thromboxane-mediated condition.
  • Patients to be treated in accordance with this aspect of the present invention can be those exhibiting symptoms of or predisposed to thromboxane-mediated conditions.
  • Symptoms of thromboxane-mediated conditions can include, without limitation, thrombosis, asthma, and those associated with anti-phospholipid syndrome.
  • Persons predisposed to thromboxane-mediated conditions are those patients currently asymptomatic, and can include those having a family history of such conditions or disorders or having had prior treatment for such conditions or disorders.
  • the treatment of pre-existing conditions relates to controlling the severity of symptoms associated with the condition. That is, symptoms can be maintained (i.e., no worsening) or improved, either substantially or wholly, with continued administration. By preventing a condition, it is intended that development of the condition or onset of the associated symptoms can be delayed or avoided, either substantially or wholly, with continued administration.
  • recombinant DNA techniques can be utilized in a gene therapy approach, particularly for treating chronic conditions that are associated with chronic thrombotic conditions or disorders, chronic CD40 ligand-mediated conditions or disorders, or chronic thromboxane-mediated conditions or disorders.
  • Gene therapy approaches for treating these conditions utilize an expression vector or plasmid that contains therein a recombinant gene encoding an inducer of a PPAR ⁇ agonist.
  • the recombinant gene can be introduced, using the expression vector, into one or more target tissues or systemically to achieve subsequent expression of the inducer of a PPAR ⁇ agonist (either constitutively, inducibly, or in a tissue specific manner).
  • the recombinant gene includes, operatively coupled to one another, an upstream promoter operable in mammalian cells, and other suitable regulatory elements (i.e., enhancer or inducer elements), a coding sequence that encodes the inducer of a PPAR ⁇ agonist, and a downstream transcription termination region.
  • Any suitable constitutive promoter or inducible promoter can be used to regulate transcription of the recombinant gene, and one of skill in the art can readily select and utilize such promoters, whether now known or hereafter developed.
  • Known recombinant techniques can be utilized to prepare the recombinant gene, transfer it into the expression vector, and administer the vector to a patient. Exemplary procedures are described in Sambrook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Press, NY (1989), which is hereby incorporated by reference in its entirety.
  • One of skill in the art can readily modify these procedures, as desired, using known variations of the procedures described therein.
  • viral vectors for delivery of either (i) a DNA molecule encoding an inducer of a PPAR ⁇ agonist such as decorin, or (ii) a DNA molecule encoding an enzyme that catalyzes formation of prostaglandin D 2 precursor, such as PGD synthases, or (iii) a DNA molecule encoding PPAR ⁇ , can be used.
  • Beta-trace (or lipocalin form or brain form) of PGD synthases and its cDNA are disclosed in White et al., “Structure and Chromosomal Localization of the Human Gene for a Brain Form of Prostaglandin D2 Synthase,” J. Biol. Chem. 267(32):23202-23208 (1992); and Genbank Accession Nos. NM — 000954, AH005144 (promoter and exon 1), M98537 (promoter and exon 1), M61901, and M98538 (exons 2-6), each of which is hereby incorporated by reference in its entirety. Although the above-referenced sequences relate to human PGD synthase (brain form), sequences are also known for other mammals such as rat and mouse.
  • the glutathione-dependent (or hematopoietic form) of PGD synthases and its cDNA are disclosed in Kanaoka et al., “Structure and Chromosomal Localization of Human and Mouse Genes for Hematopoietic Prostaglandin D Synthase: Conservation of the Ancestral Genomic Structure of Sigma-Class Glutathione S-Transferase,” Eur. J. Biochem. 267: 3315-3322 (2000); and Genbank Accession Nos. D82073, NM — 014485, and AB008825-AB008830 (exons 1-6), each of which is hereby incorporated by reference in its entirety.
  • human decorin protein and cDNA are disclosed in Vetter et al., “Human Decorin Gene: Intron-Exon Junctions and Chromosomal Localization,” Genomics 15:161-168 (1993); and Genbank Accession Nos. AH002681 and L01125-L01131, each of which is hereby incorporated by reference in its entirety. Fragments of decorin, including known isoforms thereof, can likewise be utilized in accordance with the present invention. A number of such isoforms of human decorin have previously been identified and reported in Genbank. Although the above-referenced sequences relate to human decorin, sequences are also known for other mammals such as rat.
  • the human PPAR ⁇ protein and its encoding cDNA are disclosed in Greene et al., “Isolation of the Human Peroxisome Proliferator Activated Receptor Gamma cDNA: Expression in Hematopoietic Cells and Chromosomal Mapping,” Gene Expr. 4(4-5):281-299 (1995); Elbrecht et al., “Molecular Cloning, Expression and Characterization of Human Peroxisome Proliferator Activated Receptors Gamma 1 and Gamma 2 ,” Biochem. Biophys. Res. Commun. 224(2):431-437 (1996); and Genbank Accession Nos.
  • Delivery of the expression vector or naked plasmid DNA to patient cells that are intended to be transformed can be carried out according to known procedures, which includes delivery of a composition containing a high titer of the infective transformation system or naked plasmid into the site where targeted cells reside.
  • the composition can be provided as a single administration, multiple administration, or in the form of a sustained-release DNA delivery vehicle.
  • the targeted cells/tissues include, generally and without limitation, vascular tissues, bone marrow, and structural cells. As a result, the likelihood of infecting the desired or targeted cells is significantly increased over non-targeted systemic administration.
  • a viral vector or naked (plasmid) DNA can be employed.
  • Adenovirus gene delivery vehicles can be readily prepared and utilized given the disclosure provided in Berkner, Biotechniques 6:616-627 (1988) and Rosenfeld et al., Science 252:431-434 (1991), WO 93/07283, WO 93/06223, and WO 93/07282, each of which is hereby incorporated by reference in its entirety.
  • Adeno-associated viral gene delivery vehicles can be constructed and used to deliver a gene to cells. The use of adeno-associated viral gene delivery vehicles in vitro is described in Chatterjee et al., Science 258:1485-1488 (1992); Walsh et al., Proc. Nat'l Acad. Sci.
  • Retroviral vectors which have been modified to form infective transformation systems can also be used to deliver nucleic acid encoding a desired protein or polypeptide or RNA product into a target cell.
  • One such type of retroviral vector is disclosed in U.S. Pat. No. 5,849,586 to Kriegler et al., which is hereby incorporated by reference in its entirety.
  • Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature.
  • Current methods of drug delivery via liposomes require that the liposome carrier ultimately become permeable and release the encapsulated drug at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated.
  • liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Nat'l Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989), which is hereby incorporated by reference).
  • liposomes When liposomes are endocytosed by a target cell, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in drug release.
  • the liposome membrane can be chemically modified such that an enzyme is placed as a coating on the membrane which slowly destabilizes the liposome. Since control of drug release depends on the concentration of enzyme initially placed in the membrane, there is no real effective way to modulate or alter drug release to achieve “on demand” drug delivery. The same problem exists for pH-sensitive liposomes in that as soon as the liposome vesicle comes into contact with a target cell, it will be engulfed and a drop in pH will lead to drug release.
  • This liposome delivery system can also be made to accumulate at a target organ, tissue, or cell via active targeting (e.g., by incorporating an antibody or hormone on the surface of the liposomal vehicle). This can be achieved according to known methods.
  • DNA delivery vehicles in the form of a sustained-release polymeric matrix containing the DNA to be delivered can likewise be employed to administer the DNA for purposes of gene therapy.
  • administering will modulate megakaryocytes. That is, by increasing the concentration of PPAR ⁇ present in the megakaryocytes, the megakaryocytes will be altered phenotypically. As a consequence of such treatment, it is believed that daughter platelets produced by those megakaryocytes will likewise differ phenotypically from platelets produced prior to administration of the above-identified agents. Without being bound by belief, it is believed that the platelets can differ in their composition (e.g., more PPAR ⁇ would lead to platelet dampening).
  • Such daughter platelets may also have altered expression of CD40 ligand and/or cyclooxygenase 1, which if diminished would blunt or minimize pro-inflammatory and/or pro-thrombotic responses by the platelets.
  • the platelets would be characterized by a diminished ability to activate, aggregate, and form clots.
  • the present invention also relates to several diagnostic assays.
  • One such assay relates to a method of assessing the activity of a compound as a PPAR ⁇ agonist.
  • This assay can be carried out by combining the compound with both thrombin and platelets, determining the level of CD40 ligand or thromboxane released from the platelets, and comparing the level of CD40 ligand or thromboxane released from the platelets to the level of CD40 ligand or thromboxane released from a standard, wherein deviation from the standard, or absence thereof, indicates activity of the compound as a PPAR ⁇ agonist.
  • a platelet activator e.g., thrombin, epinephrin, collagen, ADP, etc.
  • thrombin epinephrin, collagen, ADP, etc.
  • the standard includes platelets in the absence of the platelet activator, and the comparison assesses the absence of deviation between the combination and the standard.
  • the standard includes platelets, the platelet activator, and a known PPAR ⁇ agonist, and the comparison assesses the deviation between the combination and the standard.
  • the assay preferably utilizes an immunological detection procedure, using an antibody or binding portion thereof recognizing CD40 ligand or thromboxane B 2 .
  • the sample (and the standard) is contacted with the antibody or binding portion thereof and any reaction which indicates that CD40 ligand or thromboxane B 2 is present in the sample is detected.
  • Detection of antibody-CD40 ligand or antibody-thromboxane B 2 binding can be achieved using any of a variety of known detection procedures, such as enzyme-linked immunoabsorbent assay, radioimmunoassay, gel diffusion precipitin reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay, protein A immunoassay, immunoelectrophoresis assay, Western blot, immunodotblot assay, or immunoslotblot assay.
  • enzyme-linked immunoabsorbent assay such as enzyme-linked immunoabsorbent assay, radioimmunoassay, gel diffusion precipitin reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay, protein A immunoassay, immunoelectrophoresis assay, Western blot, immunodotblot assay, or immunoslotblot assay.
  • Suitable anti-CD40 ligand antibodies useful for these procedures include polyclonal antibodies and monoclonal antibodies.
  • Exemplary monoclonal antibodies include, without limitation, MK13 (Boehringer Ingelheim), 24-31 (Ancell), TRAP1 (Calbiochem Corp.), and Clone C20 (Santa Cruz).
  • Other anti-CD40 ligand antibodies or binding fragments thereof can also be used.
  • Suitable anti-thromboxane (A 2 or B 2 ) antibodies useful for these procedures include polyclonal antibodies and monoclonal antibodies.
  • Exemplary polyclonal antibodies include, without limitation, rabbit anti-TXB 2 (Novus Biologicals, Littleton, Colo.; Cayman Chem. Co., Ann Arbor, Mich.).
  • Another assay of the present invention relates to a method of diagnosing a CD40 ligand- or thromboxane-mediated condition through the use of a patient sample.
  • Suitable patient sample materials include, without limitation, blood, plasma, tissue washings, lung lavage, eye fluids, saliva, joint fluid, peritoneal fluid, stool, semen, gastric fluids, and thoracic fluids. Having thus obtained a sample, the level of PPAR ⁇ in the patient sample is detected, wherein a reduced PPAR ⁇ level indicates the presence of a CD40 ligand- or thromboxane-mediated condition (such as those listed above).
  • the assay preferably utilizes an immunological detection procedure, using an antibody or binding portion thereof recognizing PPAR ⁇ .
  • the sample is contacted with the antibody or binding portion thereof and any reaction which indicates that PPAR ⁇ is present in the sample is detected.
  • Detection of antibody-PPAR ⁇ binding can be achieved using any of a variety of known detection procedures, such as enzyme-linked immunoabsorbent assay, radioimmunoassay, gel diffusion precipitin reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay, protein A immunoassay, immunoelectrophoresis assay, Western blot, immunodotblot assay, or immunoslotblot assay.
  • Suitable anti-PPAR ⁇ antibodies useful for these procedures include polyclonal antibodies and monoclonal antibodies.
  • Exemplary monoclonal antibodies include, without limitation, the monoclonal anti-PPAR ⁇ antibody from Santa Cruz Biotechnology Inc., (Santa Cruz, Calif.).
  • Exemplary polyclonal antibodies include, without limitation, the polyclonal anti-PPAR ⁇ antiserum available from Calbiochem® Immunochemicals/EMD Biosciences (San Diego, Calif.).
  • a further assay of the present invention relates to a method of assessing the efficacy of a PPAR ⁇ agonist therapy through the use of a sample from a patient who has previously received/been administered a PPAR ⁇ agonist or an inducer of a PPAR ⁇ agonist for treating a medical condition or disorder.
  • Suitable patient sample materials include, without limitation, blood, plasma, tissue washings, lung lavage, eye fluids, saliva, joint fluid, peritoneal fluid, stool, semen, gastric fluids, and thoracic fluids.
  • the level of PPAR ⁇ in the patient sample is detected and determined, wherein an elevated PPAR ⁇ level, relative to a baseline PPAR ⁇ level for the patient prior to the administration of the agonist or inducer, indicates the efficacy of the PPAR ⁇ agonist therapy.
  • the assay preferably utilizes an immunological detection procedure, using an antibody or binding portion thereof recognizing PPAR ⁇ .
  • the sample is contacted with the antibody or binding portion thereof and any reaction which indicates that PPAR ⁇ is present in the sample is detected.
  • Detection of antibody-PPAR ⁇ binding can be achieved using any of the above-identified immunoassay, with any of the anti-PPAR ⁇ antibodies described above as reagents.
  • Meg-01 cells were purchased from the American Type Culture Collection (Rockville, Md.) and are widely used as a model of human megakaryocytes (Ogura et al., “Establishment of a Novel Human Megakaryoblastic Leukemia Cell Line, MEG-01, with Positive Philadelphia Chromosome,” Blood 66:1384-1392 (1985), which is hereby incorporated by reference in its entirety).
  • Meg-01 cells were cultured in RPMI-1640 tissue culture medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 10 mM HEPES (Sigma, St. Louis, Mo.), 2 mM L-glutamine (Invitrogen), 4.5 g/L glucose (Invitrogen) and 50 ⁇ g/ml gentamicin (Invitrogen).
  • FBS fetal bovine serum
  • HEPES human fetal bovine serum
  • 2 mM L-glutamine Invit
  • Blood samples 500 mL were collected from healthy volunteers by venipuncture into a CPDA-1 blood collection bag (Baxter Healthcare, Dearfield, Ill.). The platelet-rich plasma was obtained by centrifugation at 1,800 ⁇ g for 8 minutes and extracted into the transfer bag (Charter Medical, Winston-Salem, N.C.) at room temperature. The Pall Biomedical Purecell LRF high efficiency leukoreduction filter was used to reduce leukocytes, microaggregates and anaphylatoxin C3a. Leukocytes were removed by adherence in the filter. Platelets were washed with 0.9% saline using a COBE 2991 Blood Cell Processor (Lakewood, Colo.).
  • Meg-01 and platelet total protein was isolated using Nonidet P-40 lysis buffer containing a protease inhibitor cocktail (4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, transepoxysuccinyl-L-leucylamido (4-guanidino) butane, bestatin, leupeptin and aprotinin) (Sigma).
  • protease inhibitor cocktail 4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, transepoxysuccinyl-L-leucylamido (4-guanidino) butane, bestatin, leupeptin and aprotinin
  • Total protein was quantified with a BCA protein assay kit (Pierce, Rockford, Ill.).
  • a total of 15 ⁇ g of protein was electrophoresed on 10% denaturing polyacrylamide-stacking gels and transferred to nitrocellulose membrane (Amersham, Piscataway, N.J.) at 4° C. After blocking with 10% Blotto (PBS/0.1% Tween 20 and 10% milk) for 2 hours at room temperature, membranes were then incubated with a mouse monoclonal anti-PPAR ⁇ antibody from Santa Cruz Biotechnology (1:1000) (Santa Cruz, Calif.) or with a rabbit polyclonal anti-PPAR ⁇ antibody from Calbiochem (1:5000) (San Diego, Calif.) diluted in 2.5% Blotto for 1 hour.
  • a mouse monoclonal anti-PPAR ⁇ antibody from Santa Cruz Biotechnology (1:1000) (Santa Cruz, Calif.) or with a rabbit polyclonal anti-PPAR ⁇ antibody from Calbiochem (1:5000) (San Diego, Calif.) diluted in 2.5% Blotto for 1 hour.
  • Human bone marrow aspiration material was obtained from the hip bone of anemia patients. A drop of material about 2 mm in diameter was put onto slides and immediately spread over by cover slip and air dried for 24 hours. Smears were fixed with acetone-methanol solutions. Except for the fixation step, immunocytochemistry was performed as described (Harris and Phipps, “Prostaglandin D 2 , its Metabolite 15-d-PGJ 2 , and Peroxisome Proliferator Activated Receptor-gamma Agonists Induce Apoptosis in Transformed, but not Normal, Human T Lineage Cells,” Immunology 105:23-34 (2002), which is hereby incorporated by reference in its entirety).
  • a reaction was performed without reverse transcriptase for each cDNA synthesis and used as a negative control in the PCR. Ten ⁇ l of cDNA was used in the PCR reaction. The RT-PCR products were separated by gel electrophoresis on 1% agarose gels and stained with ethidium bromide. Adipose tissue and THP1 human monocyte cells were used as positive controls.
  • the washed platelets were re-suspended and incubated in 1 ml FACS lysis solution (FLS, BD Biosciences, Immunocytometry Systems, San Jose, Calif.) at a concentration of 1 ⁇ 10 7 /ml in 1 ⁇ FLS for 10 minutes in the dark at room temperature. After centrifugation at 500 ⁇ g for 5 minutes, the cells were permeabilized with 1 ⁇ FLS+0.2% saponin (Sigma) for 10 minutes. Samples then were incubated with 8 ⁇ g/ml monoclonal FITC-labeled anti-PPAR ⁇ antibody (BD Biosciences, San Diego, Calif.) or FITC-labeled IgG1 isotype control (BD Biosciences) for 30 minutes in the dark at room temperature.
  • washed platelets were pre-treated with PPAR ⁇ agonists for 15 minutes and were then exposed to 0.8 U/ml thrombin for 60 min at 37° C. in the presence of 200 ⁇ M fibrinogen receptor antagonist (Bachem, King of Prussia, Pa.) and 5 mM EDTA (Sigma) to prevent clotting.
  • the platelets were then stained for CD40L using a mouse IgG1 anti-human CD40L biotinylated monoclonal antibody (Ancell, Bayport, Minn.), or a mouse IgG1 isotype control antibody (Caltag, Burlingame, Calif.) followed by streptavidin conjugated to allophycocyanin (Caltag).
  • Platelets were lysed with hypotonic buffer (10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40 and 0.2 mM PMSF) and 10 ⁇ g of cell extract was incubated in each well of TransAM® PPAR ⁇ assay kit (Active Motif, Carlsbad, Calif.) and PPAR ⁇ DNA binding was determined as per manufacturers' protocol.
  • hypotonic buffer 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40 and 0.2 mM PMSF
  • Nuclear extracts of Meg-01 cells were prepared as described previously (Andrews and Faller, “A Rapid Micropreparation Technique for Extraction of DNA Binding Proteins from Limiting Numbers of Mammalian Cells,” Nucleic Acids Res. 19:2499 (1991), which is hereby incorporated by reference in its entirety). Cells were treated with 5 ⁇ M 15d-PGJ 2 , 10 ⁇ M ciglitazone, or DMSO (vehicle control) for 4 hours.
  • the cells were washed in cold PBS and then incubated on ice in hypotonic buffer (10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40 and 0.2 mM PMSF) for 10 minutes.
  • hypotonic buffer 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40 and 0.2 mM PMSF
  • the pellet was isolated carefully and re-suspended in 80 ⁇ l of hypertonic buffer (20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl 2 , 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF). After incubation on ice for 20 minutes, lysates were centrifuged for 20 seconds and the supernatant containing the nuclear protein was transferred to new tubes. Protein quantification was performed using a BCA assay kit. Platelet protein isolation was done as described for the PPAR ⁇ activity assay.
  • the consensus sequence for PPAR ⁇ (5′-CAAAACTAGGTCAAAGGTCA-3′) (SEQ ID NO: 1) was labeled with [ ⁇ -32P]ATP using T4 Polynucleotide Kinase (Life technologies). Micro Bio-Spin P-30 Tris Chromatography Columns were used to remove the unbound nucleotides (Bio-Rad).
  • Meg-01 or platelet protein extracts were incubated with binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4% glycerol, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM DTT and 0.05 mg/ml poly (dI-dC)) and 50,000 counts of labeled oligonucleotide or cold oligonucleotide for 15 minutes at room temperature.
  • Supershift experiments were completed by adding 2 ⁇ g of the anti-PPAR ⁇ antibody (Calbiochem) to the binding reaction. The samples were then run on a 4% non-denaturing polyacrylamide gel. The gel was dried on a Savant SGD 2000 gel dryer (Savant, Farmingdale, N.Y.) for 1 hour at 50° C., and exposed to film overnight.
  • Platelets were isolated as described above and cultured with buffer or with 15d-PGJ 2 or rosiglitazone (both at 20 ⁇ M) for 15 minutes at 37° C. Platelets were then activated with 0.8 U/ml thrombin or buffer and samples were taken at the 5, 10, 15, 30 and 60 minutes time points to measure human soluble CD40L and PGE 2 .
  • CD40L assays were performed with a commercially available ELISA specific for CD40L (Bender Biomedical Systems, San Bruno, Calif.). Virtually identical results were obtained using an ELISA for CD40L developed in our lab (data not shown).
  • the stable end product of platelet TXA 2 synthesis, namely TXB 2 was measured using a highly specific enzyme immunoassay from Cayman Chemical Company as per the manufacturer's directions.
  • Platelet aggregation was performed using the turbidometric method of Born (“Quantitative Investigations into the Aggregation of Blood Platelets,” J. Physiol. Lond. 162:67 (1962), which is hereby incorporated by reference in its entirety) with simultaneous measurement of ATP release using a Chrono-log Lumi-aggregometer with AGGRO/LINK for Windows Software version 5.1.6 (Chrono-log Corp., Havertown, Pa.). Blood was collected by clean venipuncture from normal donors who abstained from drugs known to affect platelet aggregation into 0.105M/sodium citrate.
  • Platelet rich plasma was prepared by centrifugation at 150 ⁇ g for 10 min at 20° C., and the platelet count adjusted to 250,000/ ⁇ l by mixing PRP and platelet poor plasma from the same donor. All experiments were performed within 3 hours of blood collection. Aggregation was performed with ADP, and the slope of aggregation and amplitude were computed using accompanying software.
  • the effects of the PPAR ⁇ agonist 15d-PGJ 2 were tested by addition of varying concentrations to PRP for 15 min before aggregation. The 15d-PGJ 2 was dissolved in DMSO, with a final concentration of DMSO in the samples of approximately 0.1%. Control experiments showed no effect of this concentration of DMSO on platelet aggregation or release.
  • clotting time (induced by clotting activator such as epinephrine or ADP) was measured in the presence or absence of a PPAR ⁇ agonist, and differences noted.
  • Meg-01 cells have been extensively used as a model of human megakaryocytes (Ogura et al., “Establishment of a Novel Human Megakaryoblastic Leukemia Cell Line, MEG-01, with Positive Philadelphia Chromosome,” Blood 66:1384-1392 (1985), which is hereby incorporated by reference in its entirety).
  • To determine whether megakaryocytes and platelets express PPAR ⁇ protein Meg-01 cells and human platelets were tested by western blot for PPAR ⁇ . Meg-01 cells and platelets were lysed and the protein analyzed for PPAR ⁇ by western blot using commercially available and widely used anti-PPAR ⁇ antibodies.
  • Meg-01 cells express PPAR ⁇ protein that co-migrated with human fat tissue PPAR ⁇ , used as a known positive control ( FIG. 1A ).
  • FIG. 1A We next evaluated highly purified human platelets for PPAR ⁇ expression.
  • Three different single donor platelets and three multiple donor pooled platelet samples were tested for PPAR ⁇ using two different anti-PPAR ⁇ antibodies ( FIGS. 1B and 1C ).
  • Human platelets express a PPAR ⁇ band, which migrated similarly to the adipose tissue PPAR ⁇ band. While the platelet preparations were highly purified (>99.99% platelets), they did contain the rare white blood cell. To determine how many white blood cells were needed to generate a PPAR ⁇ band on a western blot, experiments were completed with different numbers of white blood cells.
  • mouse platelets were similarly tested. As with the human platelets, the mouse platelets were also found to express PPAR ⁇ protein ( FIG. 1D ).
  • Meg-01 cells FIG. 2A
  • platelets FIG. 2B
  • FIG. 2D Human bone marrow was stained with Diff-Quik to identify human megakaryocytes.
  • the megakaryocyte is the largest cell of bone marrow with multi-lobated nuclei and abundant granular cytoplasm.
  • B one marrow smears were also prepared for immunocytochemistry to stain for PPAR ⁇ .
  • the right-hand panel of FIG. 2D shows staining of human megakaryocytes for PPAR ⁇ .
  • the middle panel shows no staining with an isotype control antibody (smear is lightly counterstained with hematoxylin).
  • PPAR ⁇ mRNA is Expressed in the Meg-01 Cell Line but not in Platelets
  • PPAR ⁇ mRNA in Meg-01 and platelets was examined by RT-PCR. Platelets, while enucleate, do express a range of mRNA species (Gnatenko et al., “Transcript Profiling of Human Platelets Using Microarray and Serial Analysis of Gene Expression,” Blood 101:2285-2293 (2003), which is hereby incorporated by reference in its entirety). Total RNA was isolated from Meg-01 cells and single donor or pooled platelets, and then reverse transcribed as described in the Materials and Methods section. Resulting cDNA was run in PCR reactions with control ⁇ -actin primers or primers specific for human PPAR ⁇ .
  • RNA from human adipose tissue and THP1 human monocyte cells was used as positive controls for PPAR ⁇ .
  • Meg-01 cells and the THP-1 monocytic cells express PPAR ⁇ mRNA ( FIG. 3 , lanes 6 and 7, respectively).
  • PPAR ⁇ mRNA was not present in platelet samples ( FIG. 3 , lanes 3-5).
  • Meg-01 PPAR ⁇ has DNA Binding Ability that is Enhanced by Treatment with PPAR ⁇ Ligands
  • FIG. 4A shows that Meg-01 cells have a constitutive level of active PPAR ⁇ (lane 2 ), which was increased by exposure to the natural PPAR ⁇ agonist 15d-PGJ 2 (lane 3) and to the synthetic PPAR ⁇ agonist ciglitazone (lane 4).
  • a supershift using an anti-PPAR ⁇ antibody further supported PPAR ⁇ expression in Meg-01 cells (lane 6).
  • 15d-PGJ 2 and ciglitazone increase the activation of PPAR ⁇ in Meg-01 cells.
  • Platelets have Constitutively Active PPAR ⁇ Protein that has DNA Binding Ability
  • EMSA was next performed to determine if platelet PPAR ⁇ protein can bind to the DNA PPAR response element. Lysates from three different rigorously purified platelet samples were incubated with a radioactive probe (PPAR ⁇ consensus DNA binding sequence) or cold probe ( FIG. 4B ). A discrete DNA binding band appears in the three different platelet samples (lanes 2-4). The band disappears when extracts were incubated with excess cold probe (lanes 5-7a). A supershift assay using a specific anti-PPAR ⁇ antibody was also performed and the bands shifted to a higher mass consistent with PPAR ⁇ . (lanes 8-10).
  • the ability of platelet derived PPAR ⁇ to bind its DNA consensus sequence was also measured using the TransAMTM PPAR ⁇ assay kit (Active Motif Inc.). In this method the consensus DNA sequence for PPAR ⁇ binding (or as a control mutated oligonucleotides) is plate-bound. A cell lysate is then added to the well, washed and next incubated with an enzyme-conjugated anti-PPAR ⁇ antibody that recognizes only DNA-bound PPAR ⁇ . Following substrate addition, a colored product is formed. Platelets were exposed to buffer, 15d-PGJ 2 , ciglitazone, or rosiglitazone (20 ⁇ M for all) for 2 h at 37° C. and then protein extracted.
  • PPAR ⁇ Agonists Prevent Activated Platelet Release of CD40L, TXB 2 , PGE 2 , and ATP and Inhibit Platelet Aggregation
  • platelet PPAR ⁇ played a role in attenuating platelet activation.
  • human platelets were isolated and exposed to the PPAR ⁇ ligands 15d-PGJ 2 or rosiglitazone for 15 min at 37° C. Platelets were then incubated with buffer or with thrombin, a powerful platelet activator.
  • the cells Upon platelet activation, the cells expel key bioactive mediators important for thrombosis, inflammation and vascular disease including CD40L, TXB 2 , and PGE 2 (Phipps et al., “Platelet Derived CD154 (CD40Ligand) and Febrile Responses to Transfusion,” Lancet 357:2023-2024 (2001); Best et al., “The Interrelationship Between Thromboxane Biosynthesis, Aggregation and 5-hydroxytyptamine Secretion in Human Platelets in vitro,” Thrombosis & Haemostasis 43:3840 (1980), each of which is hereby incorporated by reference in its entirety). As shown in FIGS.
  • CD40L and TXB 2 were largely prevented in platelets exposed to a naturally occurring PPAR ⁇ agonist, 15d-PGJ 2 , as well as to rosiglitazone, a synthetic PPAR ⁇ agonist.
  • the thrombin-induced release of PGE 2 was also substantially prevented in platelets exposed to 15d-PGJ 2 or rosiglitazone.
  • the thrombin induced increase in platelet surface CD40L was also prevented by the PPAR ⁇ agonists as measured by flow cytometry ( FIG. 7 ).
  • RXR retinoic receptor
  • Platelet activation was induced using epinephrine or ADP, and clotting time was assessed using a PFA-100.
  • the PFA-100 allows for an approximation of bleeding times in patients. Longer closure times equal a lower ability to form a clot.
  • the PPAR ⁇ agonist rosiglitazone (Avandia) attenuates the ability of the platelet activator epinephrine to induce a clot.
  • the PPAR ⁇ agonist 15d-PGJ2 attenuates the ability of the platelet activator ADP to induce a clot.
  • PPAR ⁇ is believed to be expressed only by nucleated cells since it is known as a transcription factor mainly located in the nucleus (Murphy and Holder, “PPAR ⁇ Agonists: Therapeutic Role in Diabetes, Inflammation and Cancer,” Trends Pharmacol Sci. 21:469-474 (2000), each of which is hereby incorporated by reference in its entirety).
  • FIG. 13 shows the basic mechanism whereby PPAR ⁇ is believed to be activated by ligand binding to form an active transcriptional complex.
  • the current models indicate that optimal DNA binding to a PPAR DNA response element occurs after ligand binding and after heterodimerization with the retinoic receptor (RXR). Note that many studies also show that some DNA binding can occur in the absence of deliberate addition of PPAR ⁇ agonist. This is likely due to the presence of low levels of endogenous PPAR ⁇ and RXR ligand in cells.
  • Megakaryocytes the precursor cell of platelets express a wide-range of mRNA encoding for a variety of bioactive mediators (Soslau et al., “Cytokine mRNA Expression in Human Platelets and a Megakaryocytic Cell Line and Cytokine Modulation of Platelet Function,” Cytokine 9:405-411 (1997), which is hereby incorporated by reference in its entirety).
  • the Meg-01 cell line was used to test for the presence of PPAR ⁇ mRNA, and these cells do express PPAR ⁇ mRNA.
  • PPAR ⁇ could interfere, including calcium or protein kinase C signaling pathways, rearrangement of the cytoskeleton during platelet activation, or docking and fusion of granules with the plasma membrane. Further studies to determine the novel PPAR ⁇ targets in platelets will be necessary to thoroughly define the mechanism of platelet inhibition by PPAR ⁇ agonists.
  • PPAR ⁇ may be involved in the differentiation and proliferation of bone marrow cells and may have additional immunologically relevant effects in erythroid, myeloid, monocytic, megakaryocytic, T and B lymphocytic, stromal and endothelial cell function.
  • 15d-PGJ 2 and the thiazolidinedione class of anti-diabetic drugs, ciglitazone and rosiglitazone play an important role in attenuating platelet activation. This was demonstrated by the ability of PPAR ⁇ agonists to block thrombin-induced platelet release of TXB 2 CD40L, and surface-associated CD40L.
  • the PPAR ⁇ agonist 15d-PGJ 2 blunted ADP-induced platelet aggregation and ATP release.
  • Platelets the most numerous, enucleate and tiny blood cells, are not only essential for clotting, but are broadly involved in inflammation and pathogenesis. Platelets contain pro-inflammatory and bioactive mediators that include transforming growth factor- ⁇ , prostaglandins, thromboxanes and CD40L.
  • TXA 2 potentiates platelet aggregation at concentrations produced by activated platelets and mediates fever and inflammation by induction of the cyclo-oxygenase-2 enzyme (Halushka et al., “Increased Platelet Thromboxane Synthesis in Diabetes Mellitus,” J. Lab. Clin. Med.
  • Platelets have the highest expression of CD40L of any human cell. Platelet released CD40L, as well as CD40L expressed on the platelet surface, could activate nearby CD40 -expressing cells.
  • CD40L is now also considered a primary platelet agonist (Prasad et al., “Soluble CD40Ligand Induces ⁇ 3 Integrin Tyrosine Phosphorylation and Triggers Platelet Activation by Outside-in Signaling,” Proc. Natl. Acad. Sci. USA 100:12367-12371 (2003), which is hereby incorporated by reference in its entirety).
  • platelets are activated by their own released CD40L through B 3 integrin binding, a decrease in CD40L by PPAR ⁇ ligands, could reduce platelet activation, including thrombosis (Prasad et al., “Soluble CD40Ligand Induces ⁇ 3 Integrin Tyrosine Phosphorylation and Triggers Platelet Activation by Outside-in Signaling,” Proc. Natl. Acad. Sci. USA 100: 12367-12371 (2003), which is hereby incorporated by reference in its entirety).
  • CD40-CD40L interaction promotes proinflammatory and proatherogenic effects in vitro and in vivo (Lutgens et al., “Both Early and Delayed Anti-CD40L Antibody Treatment Induces a Stable Plaque Phenotype,” Proc. Natl. Acad. Sci. USA 97:7464-7469 (2000), which is hereby incorporated by reference in its entirety).
  • the pathogenesis of type 1 and type 2 diabetes involves inflammation with elevated blood levels of CD40L as in atherosclerosis (Varo et al., “Elevated Plasma Levels of the Atherogenic Mediator Soluble CD40Ligand in Diabetic Patients: A Novel Target of Thiazolidinediones,” Circulation 107:2664-2669 (2003), which is hereby incorporated by reference in its entirety).
  • PPAR ⁇ -activating thiazolidinediones novel insulin-sensitizing anti-diabetic agents, have been shown to exhibit anti-inflammatory effects (Jiang et al., “PPAR-gamma Agonists Inhibit Production of Monocyte Inflammatory Cytokines,” Nature 391:82-86 (1998); Ricote et al., “The Peroxisome Proliferator-activated Receptor-gamma is a Negative Regulator of Macrophage Activation,” Nature 391:79-82 (1998), each of which is hereby incorporated by reference in its entirety).
  • the above examples surprisingly demonstrate platelet PPAR ⁇ expression and its role in tempering platelet activation, and therefore reveal a novel target for PPAR ⁇ agonists.
  • FIG. 14 the above results demonstrate that by regulating platelet activation, that PPAR ⁇ agonists reduce the ability of human platelets to release key mediators of inflammation, including thromboxanes and CD40 ligand.
  • CD40 ligand is now viewed as a key link between platelets, inflammation and thrombosis.
  • CD40 ligand levels are known to be elevated in diabetics and thus may be important in ongoing vascular injury, inflammation and the procoagulant phenotype in diabetics.
  • PPAR ⁇ is expressed by healthy and diabetic platelets, is functionally active and that PPAR ⁇ ligands modulate the ability of platelets to become activated and produce mediators of inflammation that ultimately contribute to thrombosis and vascular injury.
  • the overall biological significance of the above findings include the discovery of the direct effects of anti-diabetic PPAR ⁇ agonists on platelets that attenuate their activation, and thus may prove to be a new class of anti-thrombotic agents that will prove useful for diabetics and for others predisposed to cardiovascular disease.

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KR102578152B1 (ko) * 2020-08-31 2023-09-14 부산대학교 산학협력단 PPARγ 길항제를 유효성분으로 함유하는 저어분사료용 첨가제 조성물

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EP2037277A1 (fr) * 2007-09-11 2009-03-18 F. Hoffmann-La Roche AG Procédés d'évaluation de l'intégrité fonctionnelle et de la qualité d'un échantillon contenant des plaquettes sanguines
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WO2009058849A1 (fr) * 2007-10-29 2009-05-07 University Of Rochester Utilisation de composés électrophiles pour induire une production plaquettaire ou maintenir une fonction plaquettaire
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